Production of cotton fiber with improved fiber characteristics

ABSTRACT

Methods are disclosed to achieve an improvement in the characteristics and yield of cotton fibers. In one method, a cotton plant of the genus Gossypium in seed form or in growth stage is treated with a brassinosteroid. In another method, an ovule culture is prepared from a cotton plant of the genus Gossypium in a brassinosteroid-containing liquid medium. Cotton fibers with improved fiber characteristics are obtained from the cotton bolls of the treated plant or from the cultured ovules. Also disclosed are a method for inducing specific genes expression in a cotton plant to produce cotton fibers with improved fiber characteristics by treatment with a brassinosteroid, and a cotton plant produced by this method; as well as a novel gene capable of changing the degree of its expression found in the ovules of a cotton plant treated with a brassinosteroid, a gene capable of hybridizing with this novel gene, a recombinant plasmid containing this novel gene, a transformant containing this recombinant plasmid, and the like.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for producing cottonfibers with improved fiber characteristics such as increased fiberlength, increased fiber fineness and higher fiber strength.

BACKGROUND OF THE INVENTION

[0002] Usually, cotton fibers are produced by cultivating a cotton plantof the genus Gossypium and collecting the cotton fibers from thecapsules (cotton bolls) formed on the cotton plant. There are manyvarieties of cotton plant, from which cotton fibers with different fibercharacteristics can be obtained and used for various applicationsdepending on their fiber characteristics. Cotton fibers arecharacterized by various properties among which fiber length, fiberfineness and fiber strength are particularly important. Many previousefforts have been made to improve the characteristics of cotton fibers.Attempted improvements have been mainly focused on fiber length andfiber fineness. In particular, there has been a great demand for longerand finer cotton fibers. The variety of cotton plant known as Sea Islandis famous for desired fiber characteristics; however, this variety ofcotton plant exhibits a poor yield of cotton fibers, therefore the priceof Sea Island cotton fibers is very high. If highly yielding cottonplants with fiber characteristics equal to or better than those of SeaIsland cotton can be produced, it will be a great contribution toindustry.

[0003] The methods for improving the characteristics or yield of cottonfibers can be roughly classified into the following three categories:

[0004] 1. Variety improvement by cross breeding

[0005] This method has been utilized most widely so far. At present,almost all the cultivated varieties of cotton plant were bred by thismethod. However, much time is needed for this method, and because of alimit to the degree of variability, one cannot expect remarkableimprovements in fiber characteristics or in yield of cotton fibers.

[0006] 2. Treatment with plant hormone

[0007] Plant hormones such as auxin, gibberellin, cytokinin and ethylenehave been widely applied to field crops or horticultural products. Manyreports have hitherto been made with respect to the influence of planthormones on the fiber production of cotton plants, particularly on thefiber elongation mechanism. It is believed that fiber elongation isinduced by gibberellin or auxin but inhibited by abscisic acid (Bhardwajand Sharma, 1971; Singh and Sing, 1975; Baert et al., 1975; Dhindsa etal., 1976; Kosmidou, 1976; Babaev and Agakishiev, 1977; Bazanova, 1977;DeLangbe et al., 1978). Also Beasley and Ting [Amer. J. Bot., 60(2):130-139 (1973)] have reported that gibberellin has a promoting effect onthe fiber elongation in ovule (in vitro) whereas kinetin and abscisicacid have an inhibitory effect on the fiber elongation.

[0008] In a field test (in vivo), when non-fertilized flowers of cottonplants were treated with gibberellin just after flowering, there wasfound a promoting effect on the fiber elongation to a certain degree; inthe case of fertilized flowers, however, no significant promotion wascaused by gibberellin treatment (The Cotton Foundation Reference Book,Series Number 1, Cotton Physiology, 369, The Cotton Foundation, 1986).

[0009] As to the influence of plant hormones on the yield of cottonfibers was analyzed by MaCarty and Hedin who reported as follows: afield test on commercial plant growth regulators were conducted for aperiod of from 1986 to 1992. They found only in the field test of 1992that an increase in fiber yield was observed with a Foliar Trigger(manufactured by Westbridge Chemical Co.) containing cytokinin or withFPG-5 (manufactured by Baldridge Bio-Research, Inc.) containingcytokinin, indoleacetic acid and gibberellin; however, no significantincrease in fiber yield was observed in the other years [J. Agric. FoodChem., 42: 1355-1357 (1994)].

[0010] As described above, for the purpose of improving thecharacteristics and yield of cotton fibers, a number of studies andreports have been made on conventional plant hormones such as auxin,gibberellin, cytokinin and abscisic acid; however, no effect has beenfully confirmed yet, and it cannot be said that these plant hormones areeffective for practical use.

[0011] In recent years, much attention has been paid to brassinosteroidsas a novel group of plant hormones, and the action of these hormones onvarious plants has been studied. For the first time, Mitchell, Mandave,et al., discovered brassinolide, which is ore of the brassinosteroids,from Brassica napus pollen [Nature, 225, 1065 (1970)], and theyconfirmed that it has a remarkable effect on the cell elongation in theyoung buds of kidney bean. As described above, brassinolide is one ofthe steroid compounds with complicated structure, and many compoundswith structural similarities thereto have since been discovered fromvarious plants.

[0012] The effects of brassinosteroid when applied to cotton plants, wasreported by Luo et al. [Plant Physiology Communications, 5: 31-34(1988)] that the treatment of boll stalks with 0.01 or 1 ppmbrassinolide reduced the shedding of young bolls in a field test (invivo). However, no report has hitherto been made that thecharacteristics or yield of cotton fibers can be improved by use of anybrassinosteroid.

[0013] For callus culture (in vitro), Wang et al. [Plant PhysiologyCommunications, 28(1): 15-18 (1992)] reported that the addition of 0.01ppm brassinolide to MS medium induced the callus formation andembryogenesis in cotton plants. However, no report has hitherto beenmade that the characteristics or yield of cotton fibers can be improvedby addition of a brassinosteroid to a medium used for the ovule culturein the production of cotton fibers.

[0014] 3. Variety improvement by gene recombination technique

[0015] In recent years, gene recombinant technique has made startlingprogress, and several reports have been made on the successful varietyimprovement in cain kinds of plants (e.g., tomato, soybean) byintroduction and expression of a particular gene in these plants toconfer a desired genetic trait thereon. If a gene associated with fiberformation and elongation can be introduced into cotton plants andexpressed in large quantities, it would become possible to make aremarkable improvement in the characteristics or yield of cotton fibers.At present, however, only the following studies have been made on cottonplants: one is to improve insect resistance by introduction of a genecoding for BT toxin (Bacillus thuringiensis produced insecticidalprotein toxin), and the other is to improve herbicide (Glyphosate)resistance by introduction of a gene coding for 5-enol-pyruvilshikimicacid 3-phosphate synthetase. These attempts result in an improved yieldof cotton fibers per unit area but do not contribute to the improvementin the yield of cotton fibers per plant. The mechanism of fiberformation and elongation in cotton plants has not yet been fullyelucidated and also very little is now known as to what genes areassociated therewith.

SUMMARY OF THE INVENTION

[0016] Under these circumstances, the present inventors have intensivelystudied to improve the characteristics and yield of cotton fibers. As aresult, they have found that this problem can be solved by tint ofbrassinosteroids, and they have further found a gene associated with theformation and elongation of cotton fibers, thereby completing thepresent invention.

[0017] Thus, the present invention provides methods for producing cottonfibers with improved fiber characteristics, as well as cotton fibersproduced by these methods. One method involves tong a cotton plant ofthe genus Gossypium in seed form or in growth stage with abrassinosteroid, growing the cotton plant to form cotton bolls, andcollecting cotton fibers from the cotton bolls of the grown plant.Another method involves preparing an ovule culture from a cotton plantof the genus Gossypium in a brassinosteroid-containing liquid medium andcollecting cotton fibers from the cultured ovules.

[0018] The present invention also provides a method for inducingspecific genes expression in a cotton plant to produce cotton fiberswith improved fiber characteristics, which comprises treating the cottonplant in seed form or in growth stage with a brassinosteroid; as well asa cotton plant produced by this method, and a cotton seed produced bythis method.

[0019] The present invention further provides a novel gene derived froma cotton plant of the genus Gossypium, capable of changing the degree ofits expression by treatment with a brassinosteroid; a gene capable ofhybridizing with this novel gene; anti-sense DNA and RNA to this novelgene; a recombinant plasmid containing this novel gene; a transformantcontaining this recombinant plasmid and a transformed plant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a photograph showing the effects of brassinolidetreatment on the cotton fibers (ovules) of Ligon lintless 2 (Gossypiumhirsutum).

[0021]FIG. 2 is another photograph showing the effects of brassinolidetreatment on the cotton fibers (divided ovules) of Ligon lintless 2(Gossypium hirsutum).

[0022]FIG. 3 is a photograph showing the effects of brassinolidetreatment on the cotton fibers of Coker 312 (Gossypium hirstum).

[0023]FIG. 4 is a diagram showing the construction of plasmid pBI35S-22.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The methods for producing cotton fibers according to the presentinvention are based on the novel finding that the characteristics andyield of cotton fibers can be improved by treatment of cotton plantswith brassinosteroids.

[0025] The methods of the present invention can be applied to variousvarieties of cotton plant, which include, for example, Gossypiumhirsutum, Gossypium barbadense, Gossypium arboreum, Gossypium anomalum,Gossypium armourianum, Gossypium klotzchianum and Gossypium raimondii.

[0026] The cotton plant to be treated in the methods of the presentinvention may be in seed form or in growth stage. The growing cottonplant may be treated in whole or in part. The plant part for treatmentis not particularly limited, but it is preferably selected from thegroup consisting of flower buds, flowers, ovules, ovaries, bracts,leaves, stems, roots, boll stalks and young bolls.

[0027] The growth stage for treatment, although it is not particularlylimited, is preferably a period after flowering, more preferablyextending from the 2nd day to the 20th day after flowering.

[0028] The brassinosteroids which are used in the methods of the presentinvention include various compounds with the different steroid skeletonsas depicted below.

[0029] The compounds of the first type, such as brassinolide(2α,3α,22R,23R-tetra-hydroxy-24S-methyl-B-homo-7-oxa-5α-cholestan-6-one),dolicholide, homodolicholide, 24-epibrassinolide and 28-norbrassinolide,have a steroid skeleton of the formula:

[0030] For example, brassinolide is represented by the chemicalstructural formula:

[0031] The compounds of the second type, such as castasterone,dolichosterone, homodolichosterone, homocastasterone,28-norcastasterone, tiffasterol, teasterol, 24-epicastasterone,2-epicastasterone, 3-epicastasterone, 3,24-diepicastasterone,25-methyldolichosterone, 2-epi-25-methyldolichosterone and2,3-diepi-25-methyldolichosterone, have a steroid skeleton of theformula:

[0032] The compounds of the third type, such as 6-deoxocastasterone,6-deoxodolichosterone and 6-deoxyhomodolichosterone, have a steroidskeleton of the formula:

[0033] The brassinosteroid may be formulated into any composition form,preferably a liquid, paste, powder or granule formulation. Thebrassinosteroid concentration is not particularly limited, so long as itis, even if low, effective for the purpose of the present invention.Such a concentration is usually in the range of from 1×10⁻⁸ to 100 ppm,preferably from 1×10⁻⁶ to 50 ppm, and more preferably from 1×10⁻⁴ to 10ppm.

[0034] The composition may further contain any other plant hormone suchas auxin, gibberellin, cytokinin or abscisic acid, and if necessary, anyconventional additives such as surfactants, emulsifiers, wetting agentsand vehicles.

[0035] In the case of a liquid composition, it may be prepared bydissolving or dispersing a predetermined amount of the brassinosteroidin water. For the purpose of attaining stable effects, auxiliaryadditives such as emulsifiers, wetting agents and diluents are usuallyadded to the solution or dispersion. It is a general way to prepare aconcentrate and dilute it with water to yield a predeterminedconcentration before use.

[0036] A typical example of the concentrate is prepared from thefollowing ingredients: (1) 50% to 98% by weight, preferably 60% to 95%by weight, of a lower aliphatic alcohol such as methanol, ethanol,n-propanol, iso-propanol, n-butanol, iso-butanol or sec-butanol; (2) 1%to 25% by weight, preferably 2% to 20% by weight, of dimethylsulfoxideand an amide polar solvent such as dimethylformide (DMF),N-methylpyrrolidone (NMP) or dimethylacetamide (DMAA); (3) 1% to 25% byweight, preferably 2% to 20% by weight, of a water-soluble polymerselected from the group consisting of polyalkylene glycols such aspolyethylene glycol (PEG), polypropylene glycol and polyethylene glycol;polyvinylpyrrolidone; polyvinyl alcohol; and copolymers thereof; and (4)0.003 to 1.8 parts by weight, preferably 0.1 to 1.7 parts by weight,based on the total weight of the ingredients (1), (2) and (3) other thanwater, of a wetting agent selected from the group consisting ofpolyoxyalkylene ethers such as polyoxyethylene dialkyl ethers,polyoxyethylene alkyl allyl ethers and polyoxyethylene diallyl ether,polyoxyalkylene diesters such as polyoxyethylene dialkyl esters,polyoxyethylene alkyl allyl esters and polyoxyethylene diallyl esters;sulfonates such as sodium dinaphthylmethanesulfonate and calciumligninsulfonate dialkylsulfosuccinate.

[0037] In the case of a paste composition, it may be prepared by mixingthe above liquid composition with a base mater such as lanolin orvaseline.

[0038] In the case of a powder composition, it may be prepared by mixingthe brassinosteroid with an appropriate amount of a solid carrier suchas clay, kaoline, talc, diatomaceous earth, silica, calcium carbonate,monmolinite, bentonite, feldspar, quartz, alumina or sawdust.

[0039] In the case of a granule composition, it may be prepared bygranulating the above powder composition according to the conventionalmethods.

[0040] The composition of a brassinosteroid thus formulated is used fortreatment of a cotton plant in seed form or in growth stage by aspraying, application or immersion technique. As described above, thegrowing cotton plant may be treated in whole or in part. The plant partsfor treatment include flower buds, flowers, ovules, ovaries, bracts,leaves, stems, roots, boll stalks and young bolls.

[0041] To maintain the effects of a brassinosteroid, a paste compositionprepared by mixing the brassinosteroid with a base material such aslanolin may be applied to the boll stalks or ovaries of a cotton plant.Also, as a method for treatment of a cotton plant with more convenientprocedures for a short time, seed of a cotton plant may be immersed inthe composition containing a brassinosteroid dissolved or dispersed inan organic solvent. In general, spraying over the cotton plant in wholeis preferred. In particular, direct spraying to the ovaries of a cottonplant in fiber elongation stage between the 2nd day to the 20th dayafter flowering is still more effective.

[0042] The cotton plant thus treated is grown to form cotton bolls, fromwhich the cotton fibers with improved fiber characteristics arecollected.

[0043] The cotton fibers with improved fiber characteristics can also beobtained by ovule cultures of a cotton plant in abrassinosteroid-containing liquid medium. The ovule cultures can beprepared by the following method.

[0044] The liquid medium which can be used is not particularly limited,so long as the ovules of a cotton plant can be grown therein. Such aliquid medium is preferably selected from the group consisting ofMurashige-Skoog (MS) medium, Gamborg (B5) medium, Schenk-Hildebrandt(SH) medium, White (W) medium, Linsmaier-Skoog (LS) medium andBeasley-Ting (BT) medium, to which a brassinosteroid is added beforeuse. Particularly preferred is Beasley-Ting (BT) medium.

[0045] The amount of the brassinosteroid to be used is not particularlylimited, so long as it is a lower concentration to the medium for ovulecultures (in vitro culture system) but effective for the purpose of thepresent invention. Such a concentration is usually in the range of from1×10⁻⁸ to 100 ppm, preferably from 1×10⁻⁶ to 50 ppm, and more preferablyfrom 1×10⁻⁴ to 10 ppm.

[0046] Further, in addition to the brassinosteroid, other ingredientssuch as sugars, vitamins and plant hormones are preferably added to themedium. Examples of the plant hormone which can be used are auxin andits analogues such as indoleacetic acid (IAA), naphthaleneacetic acid(NAA), indolebutyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D);gibberellin (GA₃) and its analogues; cytokinins such as kinetin; andabscisic acid. These plant hormones may be used alone or in combination.The concentration of the plant hormone to be added is usually in therange of from 0.005 to 100.0 μM, preferably from 0.05 to 50 μM.

[0047] Because the brassinosteroids are hardly soluble in water, theyare dissolved in an organic solvent and the solution is then mixed withwater before the addition to the medium. Examples of such an organicsolvent are lower aliphatic alcohols such as methanol, ethanol andpropanol; lower aliphatic ketones such as methyl ethyl ketone and methylisobutyl ketone; and lower aliphatic ethers such as dimethyl ethers anddiethyl ethers.

[0048] The ovule cultures can be prepared by the conventional methods,except that a brassinosteroid-containing liquid medium is used.

[0049] For example, the ovules of a cotton plant are sterilized with asolution of sodium hypochlorite or the like, and the ovules thussterilized are aseptically placed on the liquid medium containing abrassinosteroid, followed by stationary incubation. The incubationtemperature is usually in the range of from 20° to 40° C., preferablyfrom 25° to 35° C. The cotton fibers with improved characteristics arecollected from the ovules thus cultured.

[0050] In the present invention, it was observed that the treatment of acotton plant in seed form or in growth stage with a brassinosteroidaccording to the above method gave an improvement in the fibercharacteristics such as an increase in fiber length. In view of thisfact, it is suggested that some particular genes of a cotton plant,which are associated with the formation and elongation of cotton fibers,change remarkably the degree of their expression by treatment withbrassinosteroids.

[0051] From the cotton plant grown by the above method, the desiredgenes capable of changing the degree of their expression can be isolatedby the differential screening method, subtraction method, differentialdisplay method or the like.

[0052] As a typical example, the gene isolation by the differentialscreening method will hereinafter be explained in detail, but such anisolation is not to be construed to limit the scope of the invention.

[0053] 1. Isolation of genes associated with formation and elongation ofcotton fibers

[0054] (1) Construction of cDNA library

[0055] First, cotton fiber are separated from the ovules of a cottonplant in the fiber elongation stage. From the separated cotton fibers,poly(A)⁺RNA is extracted by the ordinary procedures. Using the isolatedpoly(A)⁺RNA as a template, single-stranded cDNA is synthesized byreverse transcriptase with oligo(dT) primer. The single-stranded cDNA isconverted into a double-stranded cDNA by the polymerase reaction. Thedouble-stranded cDNA is inserted into an appropriate vector, with whichhost cells such as Escherichia coli are transformed, thereby obtaining acDNA library.

[0056] The poly(A)⁺RNA isolation and cDNA synthesis may also beperformed by use of a commercially available cDNA cloning kit. Thevector for cDNA library preparation is available from various commercialsources.

[0057] (2) Screening of desired genes from cDNA library

[0058] The desired genes can be obtained by the differential screeningmethod as follows. The phage plaque pattern of the cDNA library preparedby the above method is replicated onto two filters, each of which ishybridized with either ³²P-labelled cDNA probe prepared from cottonfibers in fiber elongation stage or from cotton fibers in fibernon-elongation stage. The cDNA of the desired gene can be selected bydetection of a positive hybridization signal only from the cDNA probe ofthe treated group.

[0059] The RNA isolation, cDNA synthesis, DNA digestion, ligation,transformation, hybridization and other techniques necessary forordinary gene recombination are described in the instructional manualsof commercially available enzymes used for the respective procedures orvarious text books (e.g., Molecular Cloning edited by Maniatis et al.,Cold Spring Harbor, 1989, and Current Protocols in Molecular Biologyedited by F. M. Ausubel et al., John Wiley & Sons, Inc., 1987).

[0060] The nucleotide sequence of the cloned cDNA can be determined bythe Maxam-Gilbert method or the dideoxy chain termination method, eachof which is performed by use of a commercially available kit. Thenucleotide sequence can also be automatically determined with anauto-sequencer.

[0061] If the cDNA thus analyzed does not correspond to a full-lengthprotein coding sequence, a desired cDNA clone having such a full-lengthprotein coding sequence can be obtained by another plaque hybridizationaccording to the ordinary method, or by the RACE technique.

[0062] As an actual example of the gene obtained from cotton fibers inthis manner, the nucleotide sequence and deduced amino acid sequence areshown in Sequence Listing, SEQ ID NOs: 1 and 2, respectively. This genecontains a sequence coding for a signal peptide having the ability toeffectively pass through the cell wall.

[0063] 2. Utilization of gene associated with formation and elongationof cotton fibers

[0064] The genes obtained by the above method can be utilized for massproduction of proteins associated with fiber formation and elongation incotton and other plants.

[0065] Further, the DNA sequence coding for a signal peptide may beutilized for modification of cell wall components by expression ofvarious proteins in the cell wall, and such a technique can be appliedto the breeding of a novel plant having conferred disease resistance orthe like.

[0066] For example, a gene associated with fiber formation andelongation may be ligated to an appropriate promoter, followed byintroduction into cotton or other plants, which makes it possible toincrease the content of a desired protein. In contrast, at least onepart of the anti-sense strand (i.e., sequence complementary to thecoding sequence) of the above gene may be ligated in reverse directionto an appropriate promoter, followed by introduction into a plant andthen expression of the so-called anti-sense RNA, which makes it possibleto decrease the content of a desired protein.

[0067] Further, the DNA sequence coding for a signal peptide may beligated to another gene, followed by introduction into a plant, wherebythe gene product can be allowed to effectively pass through the cellwall.

[0068] The transformation of plants can be performed by electroporationin which protoplasts are treated with electric pulses for introductionof plasmids, or by fusion between protoplasts and small cells, cells,lysosomes or the like. Other methods can also be employed, such asmicroinjection, polyethylene glycol technique or particle gun technique.

[0069] With the use of a plant virus as a vector, a desired gene canalso be introduced into a plant. A typical example of the plant virus iscauliflower mosaic virus (CaMV). For example, the introduction of adesired gene is performed as follows. First, a virus genome is insertedin a vector derived from E. coli or the like to prepare a recombinant,and a desired gene is inserted in the virus genome. The virus genomethus modified is removed from the recombinant by restrictionendonuclease and inoculated into a plant to insert the desired gene intothe plant [Hohn et al., Molecular Biology of Plant Tumors, AcademicPress, New York, 549-560 (1982), and U.S. Pat. No. 4,407,956].

[0070] Further, there is a technique using a Ti plasmid ofAgrobacterium. When a plant is infected with bacteria of the genusAgrobacterium, a part of their plasmid DNA is transferred to the plantgenome. By making use of such a property, a desired gene can also beintroduced into a plant. Upon infection, for example, Agrobacteriumtumefaciens and Agrobacterium rhizogenes induce the formation of crowngalls and the formation of hairy roots, respectively. Each of thesebacteria has a plasmid designated “Ti-plasmid” or “Ri-plasmid” havingT-DNA (transferred DNA) and vir region. The tumor formation is caused byincorporation of T-DNA into the genome of a plant, and thentranscription and translation of an oncogene present in the T-DNA in theplant cells. The vir region per se is not transferred to the plantcells, but it is essential to the transfer of T-DNA. Also, the virregion is operable even if it is on another plasmid different from theT-DNA containing plasmid [Nature, 303, 179 (1983)].

[0071] If a desired DNA is inserted in the T-DNA on the Ti- orRi-plasmid, the desired DNA can be incorporated into the plant genomeupon infection of the plant with these bacteria of the genusAgrobacterium. In this case, a portion inducing the formation of crowngalls or hairy roots is removed from the T-DNA of the Ti- or Ri-plasmidwithout deteriorating the desired transfer function, and the plasmidthus obtained can be used as a vector.

[0072] Further, various other vectors can also be used, for example,vectors such as pBI121 (manufactured by Clontech, Co.), which aredesignated “binary vectors”. In this case, the gene associated withfiber formation and elongation is ligated in sense or anti-sensedirection to an appropriate promoter, which is inserted in the binaryvector, followed by incorporation into a plant. The binary vectors haveno vir region, and the bacteria of the genus Agrobacterium to be usedfor introduction of these vectors are, therefore, required to containanother plasmid having vir region.

[0073] These vectors serve as a shuttle vector which can be amplifiednot only in the bacteria of the genus Agrobacterium but also in E. coli.Accordingly, the recombination of Ti-plasmids can also be performed withE. coli. These vectors have antibiotic-resistance genes, and thescreening of transformants can, therefore, be readily done in thetransformation of E. coli, bacteria of the genus Agrobacterium, plantsor the like. These vectors further have 35S promoter of CaMV, and thegene inserted in these vectors can, therefore, be incorporated into theplant genome and then expressed under no regulatory control.

[0074] The following will illustrate the introduction of a desired geneby the bacteria of the genus Agrobacterium into a plant and theregeneration of whole plants from the transformed cells in the case ofArabidopsis thaliana.

[0075] According to the ordinary method, seeds of Arabidopsis thalianaare sowed in MSO plate (Murashige-Skoog inorganic salts, 4.6 g; sucrose,10 g; 1000×vitamin stock solution, 1 ml/liter; pH 6.2) and asepticallycultivated. The explants of a root are used to prepare callus cultureson CIM plate (prepared by addition of 2,4-dichlorophenoxyacetic acid andkinetin to MSO plate to yield a final concentration of 0.5 μg/ml and0.05 μg/ml, respectively). A desired gene is ligated to a promoter,which is then inserted in a plasmid having kanamycin-resistance andhygromycin-resistance genes. The bacteria of the genus Agrobacteriumtransformed with the plasmid are cultured, and the cultures are dilutedand dispensed in appropriate portions in tubes. The root explants incallus form are immersed in these tubes and cocultivated on CIM platefor several days. When the bacterial strains are grown enough to beobserved by the naked eye, the root explants are sterilized and thencultivated on SIMC plate (prepared by addition of 2-ip, indoleaceticacid and claforan to MSO plate to yield a final concentration of 5μg/ml, 0.15 μg/ml and 500 μg/ml, respectively) for several days. Theseexplants are finally cultivated on SIMCS plate (prepared by addingkanamycin and hygromycin B to SIMC plate) with a repeated change to anew plate every week.

[0076] The transformed explants are continuously grown, and theappearance of calli will be observed. Because of the screening withantibiotics, the color of non-transformed explants changed to brown. Thecultivation is continued until the transformants have a size of about 5mm to form rosettes. When they take the form of a complete rosette, thebottom parts of the transformants are cut with a surgical knife so asnot to include any callus, and transplanted on RIM plate (prepared byadding indoleacetic acid to MSO plate to yield a final concentration of0.5 μg/ml). If the parts cut from the transformants contain a largecallus, the roots, even if produced, have a tendency to spread throughthe callus, and the vascular bundle may, therefore, be oftendisconnected between the roots and the rosettes. After about 8 to 10days, these parts are transplanted on a rock wool soaked with inorganicsalts medium [5 mM KNO₃, 2.5 mM K-phosphate buffer (pH 5.5), 2 mM MgSO₄,2 mM Ca(NO₃)₂, 50 μM Fe-EDTA, 1000×microelements (70 mM H₃BO₃, 14 mMMnCl₂, 0.5 mM CuSO₄, 1 mM ZnSO₄, 0.2 mM NaMoO₄, 10 mM NaCl, 0.01 mMCoCl₂) 1 ml/liter].

[0077] The plant which has come into flower and then formed siliques istransplanted in the soil soaked with inorganic salt medium, and is grownto give seeds. The seeds are sterilized, sowed in MSH plate (prepared byadding hygromycin B to MSO plate to yield a final concentration of 5U/ml), and then germinated, thereby obtaining a transformed plant.

[0078] From this transformed plant, DNA is extracted according to theordinary method. The DNA is digested with appropriate endonuclease, andsubjected to southern hybridization by use of the gene associated withfiber formation and elongation as a probe. Thus, it can be confirmedwhether transformation has occurred in the plant.

[0079] Further, from the transformants or non-transformants, RNA isextracted according to the ordinary method, and a probe is preparedwhich has a sense or anti-sense sequence of the gene associated withfiber formation and elongation. Northern hybridization using theseprobes makes it possible to examine the degree of expression for thedesired gene.

[0080] The gene associated with fiber formation and elongation can beexpressed specifically in the fiber formation process in cotton fibercells to make a contribution to fiber elongation. Thus, if thenucleotide sequence of this gene is utilized as a maker for elongationof cotton fibers, the elucidation of fiber elongation mechanism and theisolation of a gene controlling the fiber elongation can be achieved.

[0081] With the use of a desired protein necessary for fiber formationand elongation, which also serves as a marker for fiber formation andelongation, it becomes possible to establish a technique of inducingfiber formation and elongation, to elucidate the mechanism of fiberformation and elongation, and to isolate a gene controlling the fiberformation and elongation. The present invention is, therefore, alsoquite useful in the technical field of cell formation and elongation.

[0082] Further, the nucleotide sequence coding for a protein associatedwith fiber formation and elongation can be used for gene expression byartificial means such as in vitro transcription system or with amicroorganism such as E. coli to give a protein associated with fiberformation and elongation in large quantities and in pure form. Becausethe protein thus obtained is a protein associated with fiber formationand elongation, it can be used for modifying the structure of plant cellwalls and hence can be useful for the processing of plant raw materialsused in the industrial field.

[0083] It is believed that the gene of the present invention is a keygene associated with the growth stage of plant cells because it is alsoassociated with fiber formation and elongation. Therefore, for example,by use of cauliflower mosaic virus (CaMV) 35S promoter, all the organsof a plant can be brought into form change over the whole stage of itslife cycle. The use of a regulatory promoter for light, heat or woundingmakes it possible to prepare a plant capable of changing its formdepending upon the growth environment. Further, by use of an organ- ortissue-specific promoter, a form change can be caused only in particularorgans or tissues. For example, a promoter capable of causingtranscription only at the time of fiber formation can be used forcontrolling the formation of fibers and causing a modification of fibercharacteristics.

[0084] According to the present invention, an improvement can beattained in the characteristics (e.g., fiber length, fiber fineness,fiber strength) and yield of cotton fibers. The gene of the presentinvention can be used to prepare a novel variety of cotton plant havinga genetically fixed character of producing cotton fibers with improvedfiber characteristics in high yield.

[0085] The present invention will be further illustrated by thefollowing Examples, which are not to be construed to limit the scope ofthe invention.

EXAMPLE 1 Effects of Brassinolide on Fiber Characteristics in FieldCultivation

[0086] 1. Preparation of brassinosteroid-containing liquid compositions

[0087] (1) Aqueous solution containing 0.01 ppm brassinolide

[0088] First, 0.1 mg of brassinolide (manufactured by Fuji ChemicalIndustries Co., Ltd.) was dissolved in several milliliters of ethanol.Then, 10 liters of water were added to this solution to prepare anaqueous solution of brassinolide having a concentration of 0.01 ppm.

[0089] (2) Aqueous solution containing 0.1 ppm brassinolide

[0090] First, 1 mg of brassinolide (manufactured by Fuji ChemicalIndustries Co., Ltd.) was dissolved in several milliliters of ethanol.Then, 10 liters of water were added to this solution to prepare anaqueous solution of brassinolide having a concentration of 0.1 ppm.

[0091] (3) Aqueous solution containing 0.5 ppm brassinolide

[0092] First, 5 mg of brassinolide (manufactured by Fuji ChemicalIndustries Co., Ltd.) was dissolved in several milliliters of ethanol.Then, 10 liters of water were added to this solution to pa an aqueoussolution of brassinolide having a concentration of 0.5 ppm.

[0093] 2. Growth test (conducted on May to November in 1994)

[0094] Two cotton plants, Supima (Gossypium barbadense) and Ligonlintless 2 (Gossypium hirsutum) which is a mutant variety of cottonplant having little ability to form cotton fibers (both furnished by Dr.Kohel in Southern Crops Res. Lab., USDA-ARS), were used as the testmaterials. The seeds of both cotton plants were sowed in a nursery bedon May 11, 1994, and the seedlings were transplanted in a test field twoweeks after the germination. These cotton plants began flowering on Jul.15, 1994, and the spraying treatment was performed by spraying the abovebrassinolide-containing aqueous solution with a hand sprayer over theovaries and their neighborhood of the cotton plants everyday for aperiod extending from the 2nd day to the 20th day after the flowering(corresponding to the fiber elongation stage).

[0095] At the 50th day to the 60th day after flowering, the ovaries(cotton bolls) were opened. After drying, the ovules were harvested, andcotton fibers were separated from the seeds and evaluated for fibercharacteristics.

[0096] The evaluation of Supima cotton fibers was achieved by (i)measurements of fiber length, fiber strength and fiber fineness with the900 HVI system (manufactured by Spinlab Co.) and by (ii) measurements offiber length by the sorter method and of fiber strength by the Pressleymethod. The results are shown in Tables 1 and 2. TABLE 1 EFFECTS OFBRASSINOLIDE TREATMENT ON CHARACTERISTICS OF SUPIMA COTTON FIBERS ASMEASURED BY 900 HVI SYSTEM Fiber Concentration Fiber length strengthFiber fineness Agent (ppm) (inch) (g/tex) (μg/inch) Untreated 0 1.421 ±47.604 ± 4.309 ± 0.086 0.019 3.362 Brassinolide 0.01 1.463 ± 49.402 ±4.255 ± 0.067 0.024 2.268 Brassinolide 0.1 1.455 ± 50.373 ± 4.507 ±0.069 0.015 3.231 Brassinolide 0.5 1.507 ± 52.133 ± 4.548 ± 0.040 0.0192.329

[0097] TABLE 2 EFFECTS OF BRASSINOLIDE TREATMENT ON CHARACTERISTICS OFSUPIMA COTTON FIBERS AS MEASURED BY SORTER AND PRESSLEY METHODSConcentration Fiber length Fiber strength Agent (ppm) (inch) (1000Lbs/in²) Untreated 0 1.480 ± 0.042 108.050 ± 1.717 Brassinolide 0.011.503 ± 0.021 114.914 ± 5.149 Brassinolide 0.1 1.534 ± 0.026 111.783 ±2.616 Brassinolide 0.5 1.570 ± 0.031 120.150 ± 1.863

[0098] From the results of Tables 1 and 2, it is evident that the fiberlength, fiber strength and fiber fineness of cotton fibers were allremarkably increased by the spraying treatment of cotton plants withaqueous solutions of brassinolide. An increase was also observed to acertain extent in the yield of cotton fibers.

[0099] On the other hand, the evaluation of Ligon lintless 2 cottonfibers was achieved by visual observation with the naked eye. Theresults are shown in FIGS. 1 (ovules) and 2 (divided ovules).

[0100] As can be seen from FIGS. 1 and 2, the yield of cotton fibers wasincreased even in Ligon lintless 2 which is a mutant variety of cottonplant having little ability to from cotton fibers.

EXAMPLE 2

[0101] 1. Preparation of brassinosteroid-containing liquid compositions

[0102] In the formulation of each liquid composition, symbols forabbreviation have the following meanings:

[0103] BR: brassinolide

[0104] DMF: dimethylformamide

[0105] PEG 1000: polyethylene glycol (M.W., 1000)

[0106] EtOH: ethanol

[0107] Neoesterin (manufactured by Kumiai Chemical Co., Ltd.)

[0108] According to the following formulations, mixtures A, B and C fortreatment were prepared. Ingredients Amounts (1) Mixture A (blank) DMF 5g PEG 1000 5 g Neosterin (wetting agent) 10 g EtOH 80 g Total 100 g (2)Mixture B (0.05 ppm BR) BR 2.5 mg DMF 5 g PEG 1000 5 g Neosterin(wetting agent) 10 g EtOH 80 g Total 100 g (3) Mixture C (0.3 ppm BR) BR15 mg DMF 5 g PEG 1000 5 g Neosterin (wetting agent) 10 g EtOH 80 gTotal 100 g

[0109] 2. Preparation of brassinosteroid-containing liquid compositions

[0110] Using these mixtures, the following liquid compositionscontaining brassinolide at different concentrations.

[0111] (1) Liquid composition containing no brassinolide

[0112] The mixture A was 500-fold diluted with water, and this dilutionwas used.

[0113] (2) Liquid composition containing 0.05 ppm brassinolide

[0114] The mixture B was 500-fold diluted with water, and this dilutionwas used.

[0115] (3) Liquid composition containing 0.3 ppm brassinolide

[0116] The mixture C was 500-fold diluted with water, and this dilutionwas used.

[0117] 3. Growth test (conducted May to November in 1994)

[0118] In the same manner as described in Example 1, test fields andtested plants were separately provided, and the growth test wasconducted. The spraying treatment of cotton plants with differentbrassinolide-containing liquid compositions was performed in the samemanner as described in Example 1.

[0119] At the 50th day to the 60th day after flowering, the ovaries(cotton bolls) were opened. After drying, the ovules were harvested, andcotton fibers were separated from the seeds and evaluated for fibercharacteristics. The results are shown in Table 3. TABLE 3 EFFECTS OFBRASSINOLIDE TREATMENT ON CHARACTERISTICS OF SUPIMA COTTON FIBERS ASMEASURED BY 900 HVI SYSTEM Fiber Concentration Fiber length strengthFiber fineness Agent (ppm) (inch) (g/tex) (μg/inch) Untreated 0 1.411 ±46.871 ± 4.223 ± 0.082 0.022 3.125 Brassinolide 0.05 1.475 ± 50.201 ±4.497 ± 0.052 0.017 2.511 Brassinolide 0.3 1.498 ± 51.522 ± 4.538 ±0.030 0.020 2.146

[0120] From the results of Table 3, it is evident that the fiber length,fiber strength and fiber fineness of cotton fibers were all remarkablyincreased by the spraying treatment of cotton plants with liquidcompositions of brassinolide containing a surfactant and a wettingagent.

EXAMPLE 3

[0121] 1. Preparation of brassinosteroid-containing paste composition

[0122] In the formulation of each paste composition, symbols forabbreviation have the following meanings:

[0123] BR: brassinolide

[0124] NMP: N-methylpyrrolidone

[0125] EtOH: ethanol

[0126] According to the following formulations, lanolin pastes A and Bfor treatment were prepared. Ingredients Amounts (1) Lanolin paste A(blank) NMP 0.2 ml EtOH 19.8 g Dehydrated lanolin 180 g Total 200 g (2)Lanolin paste B (0.1 ppm BR) BR 20 μg NMP 0.2 ml EtOH 19.8 g Dehydratedlanolin 180 g Total 200 g

[0127] 2. Growth test (conducted on May to November in 1994)

[0128] In the same manner as described in Example 1, test fields andtested plants were separately provided, and the growth test wasconducted. The treatment of cotton plants was started by application ofthe above lanolin pastes to the boll stalks of cotton plants on the 2ndday after flowering.

[0129] At the 50th day to the 60th day after flowering, the ovaries(cotton bolls) were opened. After drying, the ovules were collected, andcotton fibers were separated from the seeds and evaluated for fibercharacteristics. The results are shown in Table 4. TABLE 4 EFFECTS OFBRASSINOLIDE TREATMENT ON CHARACTERISTICS OF SUPIMA COTTON FIBERS ASMEASURED BY 900 HVI SYSTEM Fiber Concentration Fiber length strengthFiber fineness Agent (ppm) (inch) (g/tex) (μg/inch) Untreated 0 1.412 ±48.221 ± 4.290 ± 0.067 0.021 2.175 Brassinolide 0.1 1.449 ± 49.892 ±4.501 ± 0.032 0.011 2.143

[0130] From the results of Table 4, it is evident that the fiber length,fiber strength and fiber fineness of cotton fibers were all remarkablyincreased by the application treatment of cotton plants with abrassinolide-containing paste composition.

EXAMPLE 4 Effects of Brassinolide on Fiber Length in Ovule Cultures

[0131] Two varieties of cotton plants, Ligon lintless 2 (Gossypiumhirstum) and Coker 312 (Gossypium hirstum), were cultivated in a testfield. The ovaries at the 2nd day after flowering were collected, fromwhich the sepals, bracts and petals were removed. The ovaries thusseparated were sterilized by immersion in 70% ethanol for 30 seconds,and further sterilized by immersion in an aqueous solution containing 10wt % sodium hypochlorite and 0.05 wt % Tween 20 for 20 minutes. Theovules were aseptically taken from the ovaries using a sterile pincette,and placed in an Erlenmeyer flask which had been charged with 50 ml ofliquid media formulated as shown in Table 5 and then sterilized byautoclaving, followed by suspension culture at 32° C. TABLE 5 MEDIUMFORMULATION Ingredients Formulation 1 Formulation 2 Potassiumdihydrogenphosphate  2.0 mM  2.0 mM Boric acid  0.1 mM  0.1 mM Sodiummolybdate  0.001 mM  0.001 mM Calcium chloride  3.0 mM  3.0 mM Potassiumiodide  0.0005 mM  0.0005 mM Cobalt chloride  0.0001 mM  0.0001 mMMagnesium sulfate  2.0 mM  2.0 mM Manganese sulfate  0.1 mM  0.1 mM Zincsulfate  0.03 mM  0.03 mM Copper sulfate  0.0001 mM  0.0001 mM Potassiumnitrite  50.0 mM  50.0 mM Ferric sulfate  0.03 mM  0.03 mM Disodium EDTA 0.03 mM  0.03 mM Nicotinic acid  0.004 mM  0.004 mM Pyridoxinehydrochloride  0.004 mM  0.004 mM Thiamine hydrochloride  0.004 mM 0.004 mM Myoinositol  1.0 mM  1.0 mM D-Glucose 100.0 mM 100.0 mMD-Fructose  20.0 mM  20.0 mM Indoleacetic acid  5.0 μM  5.0 μMGibberellin  0.5 μM  0.5 μM Brassinolide  1.0 μM  0 μM pH  6.8  6.8

[0132] The ovalues cultured on the medium of formulation 1 (containingbrassinolide) were referred to as the treated group, while thosecultured on the medium of formulation 2 (containing no brassinolide)were referred to as the control group.

[0133] 1. Ligon lintless 2 (Gossypium hirstum)

[0134] After culturing for 30 days, the fiber length of Ligon lintless 2cotton fibers was measured. The measurement of fiber length wasperformed as follows. First, cotton fibers were taken from the ovulesusing a finely-tapered pincette. Because the cotton fibers thus takenwere composed of intertwined filaments, they were separated into therespective filaments. The filaments were microscopically confirmed, andthe fiber length was measured with a ruler for every 100 filamentsobtained from the receptive groups. The fiber length of the filamentsfrom the ovules was 1.98±0.27 cm in the treated group and 1.70±0.19 cmin the control filed. Further, the ovules at the 30th day after theculturing were sufficiently dried at room temperature, and cotton fiberswere taken from the dried ovules, followed by measurement of fiberweight per ovule. The fiber weight was 8.85 mg in the treated group and7.02 mg in the control group.

[0135] 2. Coker 312 (Gossypium hirstum)

[0136] The effects of brassinolide were also examined with respect toCoker 312. From the flasks of both groups, the respective ovules at the14th day after the culturing were taken and compared with each other. Anincrease in the yield of cotton fibers was clearly observed by the nakedeye. The results are shown in FIG. 3.

[0137] Then, 16 ovules at the 30th day after the culturing were taken ineach group, from which cotton fibers were separated, followed bymeasurement of fiber length. The fiber leg was 3.60±0.36 cm in thetreated group and 2.60±0.28 cm in the control group, indicating that thefilaments in the treated group was about 25% longer than those in thecontrol group. In another experiment conducted in the same manner asdescribed above, the fiber length was 4.10±0.5 cm in the treated groupand 2.80±0.5 cm in the control group, indicating the filaments in thetreated group were about 30% longer than those in the control group.

[0138] In view of these results, it is clear that the fiber length andfiber yield can be both increased by addition of brassinolide to amedium for ovule cultures of cotton plants.

EXAMPLE 5 Cloning of Gene Associated with Formation of Cotton Fibers

[0139] 1. Preparation of poly(A)⁺RNA

[0140] The cotton plant, Supima (Gossypium barbadense), cultivated in afield was used as the test material. The ovules at the 5th day to the15th day after flowering (fiber elongation stage) and those at the 25thday to the 30th day after the flowering (fiber non-elongation stage)were collected, and cotton fibers were separated from the respectiveseeds. About 5 g of the cotton fibers thus obtained were immediatelyfrozen in liquid nitrogen and pulverized with a mortar in the presenceof liquid nitrogen. Then, 10 ml of 0.2 M Tris-acetate buffer forextraction [containing 5 M guanidinethiocyanate, 0.7% β-mercaptoethanol,1% polyvinylpyrrolidone (M.W., 360,000) and 0.62% sodiumN-lauroylsarcosinate, pH 8.5] was added, and the mixture was pulverizedwith a polytron homogenizer (manufactured by KINEMATICA Co.) under icecooling for 2 minutes. At that time, β-mercaptoethanol andpolyvinylpyrrolidone were added to the buffer just before use. Thepulverized mixture was centrifuged at 17,000×g for 20 minutes, and thesupernatant was collected.

[0141] The supernatant was filtered through a miracroth, and thefiltrate was gently overlaid on 1.5 ml of 5.7 M aqueous cesium chloridein a centrifuge tube, followed by centrifugation at 155,000×g for 20hours at 20° C. The supernatant was discarded, and the precipitated RNAwas collected. The precipitate was dissolved in 3 ml of 10 mM Tris-HCland 1 mM EDTA-2Na, pH 8.0 (referred to TE buffer), to which an equalvolume of a mixture of phenol, chloroform and isoamyl alcohol (volumeratio, 25:24:1) was added. The mixture was centrifuged at 17,000×g for20 minutes, and the upper aqueous layer was collected. To the aqueouslayer, a 0.1-fold volume of 3 M aqueous sodium acetate (adjusted to pH6.2 by addition of gracious acetic acid) and a 2.5-fold volume ofethanol were added, and the mixture was well agitated and allowed tostand at −20° C. overnight. Then, the mixture was centrifuged at17,000×g for 20 minutes, and the precipitate was washed with 70% ethanoland dried in vacuo.

[0142] The dry product was dissolved in 500 μl of TE buffer to give asolution of the whole RNA. This RNA solution was incubated at 65° C. for5 minutes and immediately cooled on ice, to which an equal volume of2×coupling buffer (10 mM Tris-HCl, 5 mM EDTA-2Na, 1M NaCl, 0.5% SDS, pH7.5) was added. The mixture was overlaid on an oligo-dT cellulose column(manufactured by Clontech, Co.) which had been equilibrated withequilibration buffer (10 mM Tris-HCl, 5 mM EDTA-2Na, 0.5 M NaCl, 0.5%SDS, pH 7.5). The column was washed with an about 10-fold volume of theequilibration buffer, and the poly(A)⁺RNA was eluted with elution buffer(10 mM Tris-HCl, 5 mM EDTA-2Na, pH 7.5).

[0143] To the eluate obtained, a 0.1-fold volume of the 3M aqueoussodium acetate and a 2.5-fold volume of ethanol were added, and themixture was allowed to stand at −70° C. Then, the mixture wascentrifuged at 10,000×g, and the precipitate was washed with 70% ethanoland dried in vacuo. The dry product was dissolved again in 500 μl of TEbuffer, and the purification was repeatedly conducted with an oligo-dTcellulose column. The poly(A)⁺RNA from the fiber elongation stage wasused for preparation of a cDNA library and a cDNA probe for differentialscreening, and the poly(A)⁺RNA from the fiber non-elongation stage wasused for preparation of a cDNA probe for differential screening.

[0144] 2. Preparation of cDNA library specific to fiber elongation stage

[0145] The cDNA library was prepared with ZAP-cDNA Synthesis Kit(manufactured by Stratagene Co.). The poly(A)⁺RNA from the fiberelongation stage obtained in paragraph 1 as a template, anddouble-stranded cDNA was synthesized by reverse transcriptase witholigo(dT)primer according to the method of Gubler and Hoffman et al.[Gene, 25, 263-269 (1983)].

[0146] To both ends of the cDNA obtained, EcoRI adaptors (each havingXhoI and SpeI sites in the inside) were ligated, and the ligates DNA wasdigested with XhoI. Then, the fragment was ligated between the EcoRI andXhoI sites of the λ phage vector, λ ZAPII arm, and the vector waspackaged with an in vitro packaging kit (manufactured by Stratagene Co.,GIGAPACK Gold), followed by infection into E. coli strain SURE(OD₆₆₀=0.5), which afforded a number of recombinant λ phages serving asthe cDNA library specific to the fiber elongation stage. This cDNAlibrary had a size of 5.0×10⁶.

[0147] 3. Preparation of probes

[0148] The poly(A)⁺RNA prepared from the cotton fibers at the 15th dayin the fiber elongation stage or in the fiber non-elongation stage wasused as a template, cDNA was synthesized by reverse transcriptase M-MLV(manufactured by Toyobo Co., Ltd.) with oligo(dt) primer. After thesynthesis, alkali treatment was performed to remove the poly(A)⁺RNA byhydrolysis. The cDNA thus obtained was used as a template, and a³²P-labelled probe was prepared with Random Primed DNA Labeling Kit(manufactured by USB Co.).

[0149] The ³²P-labelled probes thus prepared by the cDNA from the fiberelongation stage and by the cDNA from the fiber non-elongation stagewere used as a positive probe and as a negative probe, respectively, fordifferential screening.

[0150] 4. Screening of gene associated with fiber formulation andelongation

[0151] The above pharges constituting the cDNA library in the fiberelongation stage were infected into E. coli cells, which were grown onLB agar medium. About 50,000 pieces of pharge DNA were replicated on twonylon membranes (Hybond-N, manufactured by Amersham Co.).

[0152] The nylon membranes having replicated pharge DNA thereon weretransferred on a filter paper containing a solution for alkalidenaturation (0.5 M NaOH, 1.5 M NaCl) and allowed to stand for 4minutes. Then, the nylon membranes were transferred on a filter papercontaining a solution for neutralization (0.5 M Tris-HCl, 1.5 M NaCl, pH8.0) and allowed to stand for 5 minutes. After washing with 2×SSC (0.3 MNaCl, 0.03 M trisodium citrate), these membranes were subjected to DNAfixation with Strata-linker (manufactured by Stratagene Co.). Themembranes thus treated for DNA fixation were prehybridized inhybridization buffer [50% formamide, 0.5% SDS, 6×SSPE (3M NaCl, 0.2 MNaH₂PO₄, 20 mM EDTA-2Na, pH 7.4), 5×Denhardt solution (0.1% Ficoll, 0.1%polyvinylpyrrolidone, 0.1% bovine serum albumin), 50 μg/ml denaturedsalmon sperm DNA] at 42° C. for 3 hours, and the cDNA probes prepared inparagraph 3 were separately added to the respective membranes, followedby hybridization at 42° C. for 20 hours. After that, the membranes wereremoved and washed with solutions each containing 2×SSC, 1×SSC, 0.5×SSCor 0.1% SSC at 42° C. for 1 to 2 hours. These membranes were dried andexposed overnight to X-ray films by allowing to closely adhere thereto.

[0153] As a result, 34 positive clones capable of hybridizing morestrongly with the positive probe (from the fiber elongation stage) thanwith the negative probe (from the fiber non-elongation stage) wereselected. The analysis was conducted for one of these positive colones,which was designated KC22.

[0154] KC22 has a partial homology with the gene of soybean exhibitingbrassinosteroid-regulated protein (BRU1) [D. M. Zurek and S. D. Clouse,Plant Physiol (ROCKV) 102, 132 (1993)]; the gene coding for xyloglucantransferase of Vigna angularis, Glycine max or the like [Nishitani etal., J. Bio. Chem., 268, 25364-25368 (1993)]; and the meri-5 geneexhibiting specific expression to the apical meristem of Arabidopsis [J.I. Medford, J. S. Elmer, and H. J. Klee, Plant Cell, 3, 359-370 (1991)].The xyloglucan transferase is an enzyme catalyzing the bridge transferof xyloglucan which is the main component of plant cell walls, and itis, therefore, believed to be a key enzyme associated with cell wallelongation.

[0155] Further, Northern analysis was performed using RNA isolated fromthe brassinolide-treated group and the control group, and it was foundthat KC22 is regulated by brassinolide.

[0156] From the pharge DNA of KC22, plasmid clone pKC22 having a cDNAinsert was prepared by the in vivo excision method with ZAP-cDNASynthesis Kit (manufactured by Stratagene Co.).

[0157] First, 200 μl of a KC22-containing pharge solution was mixed with200 μl of E. coli XL1-Blue suspension and 1 μl of helper pharge R408suspension, and the mixture was incubated at 37° C. for 15 minutes, towhich 3 ml of 2×YT medium was added. Shaken cultures were grown at 37°C. for 2 hours and treated at 70° C. for 20 minutes, followed bycentrifugation at 4000×g for 10 minutes, and the supernatant wascollected.

[0158] Then, 30 μl of the supernatant was mixed with 30 μl of E. coliSOLR suspension, and the mixture was incubated at 37° C. for 15 minutesand inoculated on several microliters of LB agar medium containing 50ppm ampicillin, followed by incubation at 37° C. overnight. Thecolony-forming E. coli contained the plasmid clone pKC22 having the cDNAinsert.

[0159] The nucleotide sequence of the cDNA insert in the plasmid pKC22was determined by the dideoxy chain termination method [Messing, Methodsin Enzymol., 101, 20-78 (1983)]. The nucleotide sequence and deducedamino acid sequence are shown in the Sequence Listing, SEQ ID NOs: 1 and2, respectively, and also shown together in the Sequence Listing, SEQ IDNo: 3. These sequences correspond to the cDNA nucleotide sequence andamino acid sequence, respectively, of a gene capable of changing thedegree of its expression at the time of fiber formation and elongation.

[0160] 5. Expression of desired gene in E. coli

[0161] The transformants obtained above were suspended in 50 ml of LBmedium containing 100 μg/ml of ampicillin, and shaken cultures weregrown at 37° C. When the turbidity OD₆₆₀ of the shaken cultures became0.2, isopropyl-β-D-thiogalactopyrenosido (IPTG) was added to yield afinal concentration of 10 mM. The shake cultures were further grown at37° C. until the turbidity OD₆₆₀ became 1.0. After completion of theculturing, bacterial cells were collected by centrifugation at 1600×gfor 15 minutes. The collected bacterial cells were suspended in a 4-foldvolume of lysis buffer [50 mM Tris-HCl (pH 8.0), 1 mM EDTA-2Na, 1 μMPMSF (phenylmethylsulfonyl fluoride), 10% sucrose], to which Lysozyme(manufactured by Sigma Co.) was added to yield a final concentration of1 mg/ml, followed by allowing to stand on ice for 10 minutes. After 10minutes, Nonidet P-40 (manufactured by Sigma Co.) was added to the cellsuspension to yield of a final concentration of 1%, and the mixture wasfurther allowed to stand on ice for 10 minutes, followed bycentrifugation at 48,000×g for 1 hour. To the supernatant obtained, anequal volume of 2×Laemli sample buffer [0.125 M Tris-HCl (pH 6.8), 20%glycerol 10% β-mercaptoethanol, 6% SDS, 0.1% bromophenol blue] wasadded, and the mixture was boiled for 2 minutes, followed bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). After completion ofthe electrophoresis, the gel was stained with Coomassie brilliant blue(CBB) and decolorized with 7% acetic acid and 25% methanol. A band wasobserved near the position corresponding to the molecular weight of 39kDa as desired, and the expression of the desired gene was thusconfirmed.

[0162] 6. Preparation of Arabidopsis thaliana transformant

[0163] (1) Construction of plasmid

[0164] The nucleotide sequence of KC22 as shown by SEQ ID NO: 1 wasdigested with DraI so as to contain the whole open reading framethereof. The DraI fragment was subcloned in the SmaI site of pUC19. Thisplasmid was digested with SalI and HindIII, in which the HindIII-XhoIfragment of 35S promoter was subcloned. This clone was digested withHindIII and SacI, and the HindIII-SacI fragment was subcloned betweenthe HindIII site and the SacI site of binary vector pBI101-Hm2. Theplasmid thus obtained was designated pBI35S-22. The construction of thisplasmid is shown in FIG. 4. The transformed E. coli JM109 was designatedE. coli JM109/pBI35S-22.

[0165] (2) Introduction of plasmid into Agrobacterium

[0166] The E. coli pBI35S-22 obtained in paragraph 6(1) and the E. colistrain HB101 containing helper plasmid pRK2013 were separately culturedon LB medium containing 50 mg/l of kanamycin at 37° C. overnight, whilethe Agrobacterium strain EHA101 was cultured on LB medium containing 50mg/l of kanamycin at 37° C. over two successive nights. Then, bacterialcells were harvested by taking 1.5 ml of each of the cultures in anEppendorf tube, and then washed with LB medium. These bacterial cellswere suspended in 1 ml of LB medium, after which three kinds of bacteriawere mixed together in 100 μl portions. The mixture was plated on LBagar medium and incubated at 28° C. for ensuring the conjugationtransfer of plasmids to Agrobacterium. After 1 to 2 days, a part of themedium was scratched by means of a sterile loop, and spread over LB agarmedium containing 50 mg/l kanamycin, 20 mg/l hygromycin B and 25 mg/lchoramphenicol. The incubation was continued at 28° C. for 2 days, and asingle colony was selected. The transformant thus obtained wasdesignated EHA101/pBI35S-22.

[0167] (3) Cultivation of sterile Arabidopsis thaliana

[0168] Several dozens of seeds of Arabidopsis thaliana stainWassilewskija (hereinafter referred to as strain WS; furnished by Dr.Shinmyo in Osaka University) were placed in a 1.5-ml tube, to which 1 mlof 70% ethanol was added, and the seeds were allowed to stand for 3minutes. The seeds were immersed in a solution for sterilization (5%sodium hypochlorite, 0.02% Triton X-100) for 3 minutes, washed fivetimes with sterile water, and then sowed in MSO plate (4.6 g ofMurashige-Skoog inorganic salts, 10 g of sucrose, 1 ml/liter1000×vitamin stock solution, pH 6.2). This plate was allowed to stand at4° C. for 2 days for low-temperature treatment and then cultivated at22° C. in a plant incubator (model MLR-350HT, manufactured by SanyoElectric Co., Ltd.) under long-day conditions (16 hours light and 8hours dark) at a light intensity of 6000 lux for 21 days. To increasethe infection efficiency, the plants were aseptically pulled out andallowed to spread their roots on a fresh MSO plate, followed bycultivation for 2 days.

[0169] (4) Infection with Agrobacterium

[0170] The roots of several pieces of the above strain WS cultivated for21 days were gathered together, cut with a surgical knife to have auniform length of about 1.5 to 2.0 cm, and placed in order on CIM plate(prepared by adding 2,4-dichlorophenoxyacetic acid and kinetin to MSOplate to yield a final concentration of 0.5 μg/ml and 0.05 μg/ml,respectively). These root explants were cultivated under long-dayconditions (16 hours light and 8 hours dark) at a light intensity of3000 lux for 2 days. MS diluent (6.4 g/liter Murashige-Skoog inorganicsalts, pH 6.3) was 3-fold diluted and dispensed in 1 ml portions intotubes, in which the roots in callus form were immersed for 10 minutes.These explants were placed in order on two layers of sterile filterpapers to remove excess water, transferred on fresh CIM plate, andcocultivated for 2 days under the same conditions as described above.

[0171] (5) Sterilization

[0172] The explants grown to a degree enough to observe the respectivebacterial strains with the naked eye were placed in a solution forsterilization (prepared by adding claforan to MS diluent to yield afinal concentration of 200 μg/ml), followed by washing with gentleshaking for 60 minutes. After five repetitions of this operation, theseexplants were placed on a sterile filter paper to remove water, placedin order on SIMC plate (prepared by adding 2-ip, IAA and claforan to MSOplate to yield of a final concentration of 5 μg/ml, 0.15 μg/ml and 500μg/ml, respectively), and cultivated under long-day conditions (16 hourslight and 8 hours dark) at a light intensity of 6000 lux for 2 days.

[0173] (6) Selection of transformed plants

[0174] The above explants cultivated for 2 days were transplanted onSIMCS plate (prepared by adding hygromycin B to SIMC plate to yield afinal concentration of 4.6 U/ml) and cultivated under long-dayconditions (16 hours light and 8 hours dark) at a light intensity of6000 lux. Thereafter, these explants were transplanted on fresh SIMCSplate every week. The transformed explants were continuously grown tobecome dome-shaped swollen calli, while the color of non-transformantschanged to brown. The calli of the transformants exhibited green colorafter about 2 weeks. After about 1 month, leaves were formed and thenbecame rosettes.

[0175] (7) Regeneration of transformed plants

[0176] The bottom parts of the plants in rosette form were cut with arazor or a surgical knife so as not to include any callus, and slightlyinserted into RIM plate as if they were placed thereon. After 8 to 10days, the plant having several roots of about 1 to 2 cm in length wastransplanted with a pincette in a mini-pot of rock wool (manufactured byNITTO BOSEKI CO., LTD) soaked with inorganic salts medium [5 mM KNO₃,2.5 mM K-phosphate buffer (pH 5.5), 2 mM MgSO₄, 2 mM Ca(NO₃)₂, 50 μMFe-EDTA, 1000×microelements (70 mM H₃BO₃, 14 mM MnCl₂, 0.5 mM CuSO₄, 1mM ZnSO₄, 0.2 mM NaMoO₄, 10 mM NaCl, 0.01 mM CoCl₂) 1 ml/liter], andcultivated. After flowering and podding, these plants were transplantedin the soil which was prepared by mixing pearlite and vermiculite(manufactured by TES Co.) at a ratio of 1:1 and soaking in inorganicsalts medium. After about 1 month, a few hundred of seeds per plant wereobtained. These seeds are hereinafter referred to as T1 seeds.

[0177] (8) Acquisition of antibiotic-resistant strains

[0178] About one hundred T1 seeds were sterilized by the same method asdescribed in paragraph 6(3), and then sowed in MSH plate. HygromycinB-resistant strains were germinated at a ratio of approximately 3:1.

[0179] 7. DNA extraction and southern hybridization

[0180] The above germinated T1 seeds were transplanted with a pincettein a mini-pot of rock wool soaked with inorganic salts medium, andcultivated at 22° C. under long-day conditions (16 hours light and 8hours dark) at a light intensity of 6000 lux. After 2 weeks, the aerialparts of the plants were cut with a surgical knife as if the surface ofthe rock wool was smoothed with a knife, and immediately frozen withliquid nitrogen. The frozen aerial parts were finely pulverized with amortar in the presence of liquid nitrogen, to which 3 ml of DNAextraction buffer [200 mM Tris-HCl (pH 8.0), 100 mM EDTA-2Na, 1% sodiumN-lauroylsarcosinate, 100 μg/ml proteinase K] was added, and the mixturewas well agitated and then incubated at 60° C. for 1 hour, followed bycentrifugation at 10,000×g for 10 minutes. The supernatant was filteredthrough a miracloth, and the filtrate was transferred in a new tube.After three extractions with a mixture of phenol, chloroform and isoamylalcohol (25:24:1), ethanol precipitation was performed. The precipitatewas dissolved in TE buffer. From about 2.0 g of the plants, 20 μg ofgenomic DNA was obtained. Each 1 μg of genomic DNA was digested withEcoRI and HIndIII, and the DNA fragments were subjected to 1% agaroseelectrophoresis and southern hybridization.

[0181] In the same manner as described, the non-transformed seeds of theWS strain were germinated and grown, after which DNA isolated from theplants was digested with EcoRI and HindIII, and the DNA fragments weresubjected to 1% agarose gel electrophoresis and souther hybridization.As the probe for hybridization, pKC22 was used.

[0182] The southern hybridization was performed according to the methoddescribed in Molecular Cloning, A Laboratory Manual, ch. 9, pp. 31-58(Cold Spring Harbor, 1989). That is, each DNA sample was subjected to 1%agarose gel electrophoresis, followed by alkali denaturation andovernight southern blotting on a nylon membrane (Hybond-N, manufacturedby Amersham Co.). The membrane was irradiated with an UVtrans-illuminator (254 nm) for 3 minutes to cause DNA fixation. Thismembrane was prehybridized in 5 ml of prehybridization buffer[5×Denhardt solution, 6×SSC, 0.1% SDS, 10 μg/ml salmon sperm DNA] at 50°C. for 2 hours, followed by hybridization with a probe at 50° C.overnight. The membrane was washed twice with a washing solutioncontaining 2×SSC and 0.1% SDS at room temperature for 10 minutes andthen twice with the same solution at 50° C. for 30 minutes. After themembrane was dried, autoradiograms were prepared by exposing themembrane to an X-ray film (manufactured by Eastman Kodak Co.) in acassett to at −80° C. overnight. Comparison of signal patterns detectedby southern hybridization was made among: (i) the non-transformants;(ii) the transformants having pKC22; and (iii) the transformants havingonly the vector.

[0183] Specific signals from the transformants (ii) were observed atpositions of about 1.6 and 0.7 kbp for the EcoRI-digested sample and ata position of about 6 kbp for the HindIII-digested sample, in additionto endogenous signals common to (i), (ii) and (iii), indicating that thedesired gene was incorporated in the transformants (ii).

What is claimed is:
 1. A method for producing cotton fibers withimproved fiber characteristics, which comprises treating a cotton plantof the genus Gossypium in seed form or in growth stage with abrassinosteroid, growing the cotton plant to form cotton bolls, andcollecting cotton fibers from the cotton bolls of the plant.
 2. Themethod according to claim 1 , wherein the cotton plant of the genusGossypium is selected from the group consisting of Gossypium hirsutum,Gossypium barbadense, Gossypium arboreum, Gossypium anomalum, Gossypiumarmourianum, Gossypium klotzchianum and Gossypium raimondii.
 3. Themethod according to claim 1 , wherein the seeds of the cotton plant aretreated with a brassinosteroid.
 4. The method according to claim 1 ,wherein the growing cotton plant is treated in whole with abrassinosteroid.
 5. The method according to claim 1 , wherein thegrowing cotton plant is treated in part with a brassinosteroid.
 6. Themethod according to claim 5 , wherein the part of the cotton plant to betreated is selected from the group consisting of flower buds, flowers,ovules, ovaries, bracts, leaves, stems, roots, boll stalks and youngbolls.
 7. The method according to claim 6 , wherein the ovaries of thecotton plant after flowering are treated with a brassinosteroid.
 8. Themethod according to claim 1 , wherein the brassinosteroid is selectedfrom the group consisting of brassinolide, dolicholide, homodolicholide,24-epibrassinolide, 28-norbrassinolide, castasterone, dolichosterone,homodolichosterone, homocastasterone, 28-norcastasterone, tiffasterol,teasterol, 24-epicastasterone, 2-epicastasterone, 3-epicastasterone,3,24-diepicastasterone, 25-methyldolichosterone,2-epi-25-methyldolichosterone, 2,3-diepi-25-methyldolichosterone,6-deoxocastasterone, 6-deoxodolichosterone and6-deoxyhomodolichosterone.
 9. The method according to claim 1 , whereinthe brassinosteroid is formulated into a liquid, paste, powder orgranule composition.
 10. The method according to claim 9 , wherein thebrassinosteroid is contained in the composition at a concentration of1×10⁻⁸ to 100 ppm.
 11. Cotton fibers produced by the method of claim 1 .12. A method for producing cotton fibers with improved fibercharacteristics, which comprises preparing an ovule culture from acotton plant of the genus Gossypium in a brassinosteroid-containingliquid medium and collecting cotton fibers from the cultured ovules. 13.The method according to claim 12 , wherein the cotton plant of the genusGossypium is selected from the group consisting of Gossypium hirsutum,Gossypium barbadense, Gossypium arboreum, Gossypium anomalum, Gossypiumarmourianum, Gossypium klotzchianum and Gossypium raimondii.
 14. Themethod according to claim 12 , wherein the brassinosteroid is selectedfrom the group consisting of brassinolide, dolicholide, homodolicholide,24-epibrassinolide, 28-norbrassinolide, castasterone, dolichosterone,homodolichosterone, homocastasterone, 28-norcastasterone, tiffasterol,teasterol, 24-epicastasterone, 2-epicastasterone, 3-epicastasterone,3,24-diepicastasterone, 25-methyldolichosterone,2-epi-25-methyldolichosterone, 2,3-diepi-25-methyldolichosterone,6-deoxocastasterone, 6-deoxodolichosterone and6-deoxyhomodolichosterone.
 15. The method according to claim 12 ,wherein the liquid medium is selected from the group consisting ofMurashige-Skoog (MS) medium, Gamborg (B5) medium, Schenk-Hildebrandt(SH) medium, White (W) medium, Linsmaier-Skoog (LS) medium andBeasley-Ting (BT) medium.
 16. The method according to claim 12 , whereinthe brassinosteroid is contained in the liquid medium at a concentrationof 1×10⁻⁸ to 100 ppm.
 17. Cotton fibers produced by the method of claim12 .
 18. A method for inducing specific genes expression in a cottonplant to produce cotton fibers with improved fiber characteristics whichcomprises treating the cotton plant in seed form or in growth stage witha brassinosteroid.
 19. The method according to claim 18 , wherein thecotton plant of the genus Gossypium is selected from the groupconsisting of Gossypium hirsutum, Gossypium barbadense, Gossypiumarboreum, Gossypium anomalum, Gossypium armourianum, Gossypiumklotzchianum and Gossypium raimondii.
 20. The method according to claim18 , wherein the brassinosteroid is selected from the group consistingof brassinolide, dolicholide, homodolicholide, 24-epibrassinolide,28-norbrassinolide, castasterone, dolichosterone, homodolichosterone,homocastasterone, 28-norcastasterone, tiffasterol, teasterol,24-epicastasterone, 2-epicastasterone, 3-epicastasterone,3,24-diepicastasterone, 25-methyldolichosterone,2-epi-25-methyldolichosterone, 2,3-diepi-25-methyldolichosterone,6-deoxocastasterone, 6-deoxodolichosterone and6-deoxyhomodolichosterone.
 21. The method according to claim 18 ,wherein the brassinosteroid is formulated into a liquid, paste, powderor granule composition.
 22. The method according to claim 21 , whereinthe brassinosteroid is contained in the composition at a concentrationof 1×10⁻⁸ to 100 ppm.
 23. The method according to claim 18 , wherein atleast one of the specific genes comprises the nucleotide sequence of SEQID NO:
 1. 24. A cotton plant produced by the method of claim 18 .
 25. Acotton seed produced by the method of claim 18 .
 26. A gene derived froma cotton plant of the genus Gossypium, capable of changing the degree ofits expression by treatment with a brassinosteroid.
 27. The geneaccording to claim 26 , which is derived from cotton fibers and ovulesof the cotton plant.
 28. The gene according to claim 26 , having thenucleotide sequence of SEQ ID NO:
 1. 29. The gene according to claim 26, coding for the amino acid sequence of SEQ ID NO:
 2. 30. A geneobtained from the gene of claim 26 by substitution or deletion in partor by insertion or addition of another gene.
 31. A gene capable ofhybridizing with the gene of claim 26 .
 32. An anti-sense DNA to thegene of claim 26 .
 33. An anti-sense RNA to the gene of claim 26 .
 34. Arecombinant plasmid containing a gene derived from a cotton plant of thegenus Gossypium, capable of changing the degree of its expression bytreatment with a brassinosteroid.
 35. A transformant containing therecombinant plasmid which contains a gene derived from a cotton plant ofthe genus Gossypium, capable of changing the degree of its expression bytreatment with a brassinosteroid.
 36. A recombinant plasmid containing agene which is derived from a cotton plant and has the nucleotidesequence of SEQ ID NO:
 1. 37. A transformant containing the recombinantplasmid of claim 36 .
 38. A recombinant plasmid containing a DNAfragment coding for the amino acid sequence of SEQ ID NO:
 2. 39. Atransformant containing the recombinant plasmid of claim 38 .
 40. Amicroorganism transformed with a recombinant plasmid containing a genederived from a cotton plant of the genus Gossypium, capable of changingthe degree of its expression by treatment with a brassinosteroid. 41.The transformed microorganism according to claim 40 , wherein themicroorganism to be transformed is selected from the group consisting ofEscherichia coli and bacteria of the genus Agrobacterium.
 42. A planttransformed with a recombinant plasmid containing a gene derived from acotton plant of the genus Gossypium, capable of changing the degree ofits expression by treatment with a brassinosteroid.
 43. The transformedplant according to claim 42 , wherein the plant to be transformed isselected from the group consisting of Arabidopsis thaliana, cotton andtobacco.